Biosensor electrode mediators for regeneration of cofactors

ABSTRACT

The present invention is based on the discovery of NAD +  and NADP +  mediator compounds that do not bind irreversibly to thiol groups in the active sites of intracellular dehydrogenase enzymes. Such mediator compounds avoid a common mode of enzyme inhibition. The mediators can therefore increase the stability and reliability of the electrical response in amperometric electrodes constructed from NAD- or NADP-dependent enzymes.

BACKGROUND OF THE INVENTION

[0001] The invention is in the general field of electrodes foramperometric biosensors. More specifically, the invention is in thefield of compounds for use as mediators for the recycling of cofactorsused in these electrodes.

[0002] NAD- and NADP-dependent enzymes are of great interest insofar asmany have substrates of clinical value, such as glucose,D-3-hydroxybutyrate, lactate, ethanol, and cholesterol. Amperometricelectrodes for detection of these substrates and other analytes can bedesigned by incorporating this class of enzymes and establishingelectrical communication with the electrode via the mediated oxidationof the reduced cofactors NADH and NADPH.

[0003] NAD- and NADP-dependent enzymes are generally intracellularoxidoreductases (EC 1.x.x.x). The oxidoreductases are further classifiedaccording to the identity of the donor group of a substrate upon whichthey act. For example, oxidoreductases acting on a CH—OH group within asubstrate are classified as EC 1.1.x.x whereas those acting on analdehyde or keto-group of a substrate are classified as EC 1.2.x.x. Someimportant analytes (e.g., glucose, D-3-hydroxybutyrate, lactate,ethanol, and cholesterol) are substrates of the EC 1.1.x.x enzymes.

[0004] The category of oxidoreductases is also broken down according tothe type of acceptor utilized by the enzyme. The enzymes of relevance tothe present invention have NAD⁺ or NADP⁺ as acceptors, and areclassified as EC 1.x.1.x. These enzymes generally possess sulphydrylgroups within their active sites and hence can be irreversibly inhibitedby thiol-reactive reagents such as iodoacetate. An irreversibleinhibitor forms a stable compound, often through the formation of acovalent bond with a particular amino acid residue (e.g., cysteine, orCys) that is essential for enzymatic activity. For example,glyceraldehyde-3-P dehydrogenase (EC 1.2.1.9) is stoichiometricallyalkylated by iodoacetate at Cys₁₄₉ with concomitant loss of catalyticactivity. In addition, the enzymes glucose dehydrogenase,D-3-hydroxybutyrate dehydrogenase (HBDH), and lactate dehydrogenase areknown to be irreversibly inhibited by thiol reagents. Thus, in seekingto develop stable biosensors containing NAD- or NADP-dependentdehydrogenases, avoidance of compounds that are reactive toward thiolsis imperative, as they can act as enzyme inhibitors.

SUMMARY OF THE INVENTION

[0005] The present invention is based on the discovery of NAD⁺ and NADP⁺mediator compounds that do not bind irreversibly to thiol groups in theactive sites of intracellular dehydrogenase enzymes. Such mediatorcompounds avoid a common mode of enzyme inhibition. The mediators cantherefore increase the stability and reliability of the electricalresponse in amperometric electrodes constructed from NAD- orNADP-dependent enzymes.

[0006] In one embodiment, the invention features a test element for anamperometric biosensor. The element includes an electrode, which hastest reagents distributed on it. The test reagents include anicotinamide cofactor-dependent enzyme, a nicotinamide cofactor, and amediator compound having one of the formulae:

[0007] or a metal complex or chelate thereof,

[0008] where X and Y can independently be oxygen, sulphur, CR³R⁴, NR³,or NR³R⁴⁻; R₁ and R₂ can independently be a substituted or unsubstitutedaromatic or heteroaromatic group; and R³ and R⁴ can independently be ahydrogen atom, a hydroxyl group or a substituted or unsubstituted alkyl,aryl, heteroaryl, amino, alkoxyl, or aryloxyl group. In some cases,either X or Y can be the functional group CZ¹Z², where Z¹ and Z² areelectron withdrawing groups.

[0009] Any alkyl group, unless otherwise specified, may be linear orbranched and may contain up to 12, preferably up to 6, and especially upto 4 carbon atoms. Preferred alkyl groups are methyl, ethyl, propyl andbutyl. When an alkyl moiety forms part of another group, for example thealkyl moiety of an alkoxyl group, it is preferred that it contains up to6, especially to 4, carbon atoms. Preferred alkyl moieties are methyland ethyl.

[0010] An aromatic or aryl group may be any aromatic hydrocarbon groupand may contain from 6 to 24, preferably 6 to 18, more preferably 6 to16, and especially 6 to 14, carbon atoms. Preferred aryl groups includephenyl, naphthyl, anthryl, phenanthryl and pyryl groups especially aphenyl or naphthyl, and particularly a phenyl group. When an aryl moietyforms part of another group, for example, the aryl moiety of an aryloxylgroup, it is preferred that it is a phenyl, naphthyl, anthryl,phenanthryl or pyryl, especially phenyl or naphthyl, and particularly aphenyl, moeity.

[0011] A heteroaromatic or heteraryl group may be any aromaticmonocyclic or polycyclic ring system, which contains at least oneheteroatom. Preferably, a heteroaryl group is a 5 to 18-membered,particularly a 5- to 14-membered, and especially a 5- to 10-membered,aromatic ring system containing at least one heteroatom selected fromoxygen, sulphur and nitrogen atoms. 5- and 6-membered heteroaryl groups,especially 6-membered groups, are particularly preferred. Heteroarylgroups containing at least one nitrogen atom are especially preferred.Preferred heteroaryl groups include pyridyl, pyrylium, thiopyrylium,pyrrolyl, furyl, thienyl, indolinyl, isoindolinyl, indolizinyl,imidazolyl, pyridonyl, pyronyl, pyrimidinyl, pyrazinyl, oxazolyl,thiazolyl, purinyl, quinolinyl, isoquinolinyl, quinoxalinyl,pyridazinyl, benzofuranyl, benzoxazolyl and acridinyl groups.

[0012] When any of the foregoing substituents are designated as beingsubstituted, the substituent groups which may be present may be any oneor more of those customarily employed in the development of compoundsfor use in electrochemical reactions and/or the modification of suchcompounds to influence their structure/activity, solubility, stability,mediating ability, formal potential (E°) or other property. Specificexamples of such substituents include, for example, halogen atoms, oxo,nitro, cyano, hydroxyl, cycloalkyl, alkyl, haloalkyl, alkoxy,haloalkoxy, amino, alkylamino, dialkylamino, formyl, alkoxycarbonyl,carboxyl, alkanoyl, alkylthio, alkylsulphinyl, alkylsulphonyl,arylsulphinyl, arylsulphonyl, carbamoyl, alkylamido, aryl or aryloxygroups. When any of the foregoing substituents represents or contains analkyl substituent group, this may be linear or branched and may containup to 12, preferably up to 6, and especially up to 4, carbon atoms. Acycloalkyl group may contain from 3 to 8, preferably from 3 to 6, carbonatoms. An aryl group or moiety may contain from 6 to 10 carbon atoms,phenyl groups being especially preferred. A halogen atom may be afluorine, chlorine, bromine or iodine atom and any group which containsa halo moiety, such as a haloalkyl group, may thus contain any one ormore of these halogen atoms.

[0013] An electron withdrawing group may be any group, which forms astable methylene group CZ¹Z² Such electron withdrawing groups mayinclude halogen atoms, nitro, cyano, formyl, alkanoyl, carboxyl andsulphonic acid groups.

[0014] Preferably, X and Y are both oxygen atoms.

[0015] It is also preferred that R₁ and R₂ are independently selectedfrom phenyl, naphtuyl, pyridyl and pyrrolyl groups with pyridyl groupsbeing especially preferred. The term “pyridyl group” also includes theN-oxide thereof as well as pyridinium and N-substituted pyridiniumgroups.

[0016] Preferably, R₁ and R₂ are unsubstituted or substituted only byone or more, preferably one or two, alkyl groups, especially methylgroups. It is especially preferred that R₁ and R₂ are unsubstituted.

[0017] R₃ and R₄, if present, are preferably independently selected fromhydrogen atoms and alkyl groups.

[0018] Metal complex and chelates include complexes and chelates withtransition metals, especially first-, second- and third-row transitionelements such as ruthenium, chromium, cobalt, iron, nickel and rhenium,with ruthenium being particularly preferred. Other groups such as4-vinyl-4′-methyl-2,2′-bipridyl (v-bpy) and bipyridyl (bpy) groups mayalso be included in such complexes and chelates as parts of a complexmetal ion. Typically, such complexes and chelates will form as a resultof heteroatoms in R₁ and R₂ coordinating with a metal ion or metal ioncomplex.

[0019] The test reagents can be deposited on the electrode in one ormore ink-based layers. The test reagents can be screen-printed onto theworking electrode in a single layer.

[0020] The element can be an amperometric dry-strip sensor that includesan elongated, electrically insulating carrier having a pair oflongitudinal, substantially parallel electrically conducting tracksthereupon, and a pair of electrodes. The electrodes can each beelectrically connected to a different one of the tracks; one of theelectrodes can be a reference/counter electrode, while another electrodecan be a working electrode. The element can also include a dummyelectrode. Further, the element can include a membrane positioned tofilter samples prior to their introduction onto the electrodes.

[0021] The sensor can additionally include a supporting strip ofelectrically insulating carrier material (e.g., a synthetic polymer suchas polyvinyl chloride, or a blend of synthetic polymers).

[0022] The mediator compound can be a quinone. Examples of suitablequinones include 1,10-phenanthroline quinone, 1,7-phenanthrolinequinone, and 4,7-phenanthroline quinone.

[0023] In another embodiment, the invention features an electrode stripfor an amperometric sensor having a readout. The strip includes asupport adapted for releasable attachment to the readout, a firstconductor extending along the support and comprising a conductiveelement for connection to the readout: a working electrode in contactwith the first conductor and positioned to contact a sample mixture: asecond conductor extending along the support and comprising a conductiveelement for connection to the readout: and a reference/counter electrodein contact with the second conductor and positioned to contact thesample and the second conductor. The active electrode of the stripincludes a mediator compound having one of the formulae:

[0024] wherein X, Y, R₁ and R₂ are as previously defined.

[0025] Still another embodiment of the invention features a method formediating electron transfer between an electrode and a nicotinamidecofactor. The method includes the steps of using a mediator compound inthe presence of a nicotinamide cofactor-dependent enzyme, where themediator compound is a quinoid compound that is incapable of bindingirreversibly to the thiol groups. The mediator compound can, forexample, have reactive unsaturated bonds in adjacent aromatic ring.Suitable mediator compounds include those having the formulae:

[0026] wherein X, Y, R₁ and R₂ are as previously defined.

[0027] For example, the mediator compound can be 1,10-phenanthrolinequinone, 1,7-phenanthroline quinone, or 4,7-phenanthroline quinone.

[0028] In yet another embodiment, the invention features a printing ink.The ink includes a nicotinamide cofactor-dependent enzyme, anicotinamide cofactor, and a mediator compound having one of theformulae:

[0029] wherein X, Y, R¹, and R² are as previously defined.

[0030] For example, the mediator compound can be 1,10-phenanthrolinequinone, 1,7-phenanthroline quinone, or 4,7-phenanthroline quinone. Theenzyme can be, for example, alcohol dehydrogenase, lactatedehydrogenase, 3-hydroxybutyrate dehydrogenase, glucose-6-phosphatedehydrogenase, glucose dehydrogenase, formaldehyde dehydrogenase, malatedehydrolenase, or 3-hydroxysteroid dehydrogenase.

[0031] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described below. All publications, patentapplications, patents, technical manuals, and other references mentionedherein are incorporated by reference in their entirety. In case ofconflict, the present application, including definitions, will control.In addition, the materials, methods, and examples are illustrative onlyand not intended to be limiting.

[0032] An advantage of the new mediators is their non-reactivity withrespect to active-site thiol groups in enzymes. This improves thestability and the shelf life of biosensor electrodes to an unexpecteddegree. Also as a result of this stability, the enzyme and mediator canbe incorporated together in a printing ink or dosing solution tofacilitate construction of the biosensors. The use of a mediator that isnot an irreversible inhibitor of the enzyme will result in the retentionof a large proportion of enzyme activity during the biosensormanufacture. NAD- and NADP-dependent dehydrogenase enzymes are generallyexpensive and labile and improvement of their stability is thereforehighly desirable.

[0033] Advantageously, the compounds disclosed herein can also be usedas mediators to the cofactors NADH and NADPH coupled with a wide rangeof NAD- or NADP-dependent enzymes; as labels for antigens or antibodiesin immunochemical procedures; and in other applications in the field ofelectrochemistry and bioelectrochemistry. The mediators require lowoxidation potentials for re-oxidation following the reaction with NADHor NADPH. This is of particular advantage when testing in whole blood,in which the potential for interference from exogenous electroactivespecies (e.g., ascorbic acid, uric acid) is particularly high. The lowpotential can be advantageous because it can obviate the need for adummy electrode to remove electroactive species in the sample. Also, theoxidized native form of the mediator can decrease the background currentthat would be present with a reduced mediator.

[0034] Other features and advantages of the invention will be apparentfrom the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is an exploded view of an electrode strip according to oneembodiment of the invention.

[0036]FIG. 2 is a representation of an assembled electrode strip.

[0037]FIG. 3 is a graphical plot of current in μA against NADHconcentration in mM for printed electrodes containing1,10-phenanthroline quinone.

[0038]FIG. 4 is a graphical plot of current in μA against NADHconcentration in mM for printed electrodes containing Meldola's Blue.

[0039]FIG. 5 is a bar chart displaying residual enzyme activity (i.e.,as a percentage of the initial activity) after incubation of HBDH withvarious mediators.

[0040]FIG. 6 is a graphical plot of current in μA againstD-3-hydroxybutyrate concentration in mM for printed electrodescontaining 1,10-phenanthroline quinone, D-3-hydroxybutyratedehydrogenase and NAD⁺ tested after 4, 14 and 26 weeks.

[0041]FIG. 7 is a graphical plot of current in μA againstD-3-hydroxybutyrate concentration in mM for printed electrodescontaining Meldola's Blue, D-3-hydroxybutyrate dehydrogenase, and NAD⁺tested after 2 and 14 weeks, respectively.

[0042]FIG. 8 is a graphical plot of calibrated response to glucose inwhole blood for printed electrodes containing 1,10-phenanthrolinequinone, glucose dehydrogenase, and NAD⁺.

DETAILED DESCRIPTION OF THE INVENTION

[0043] A class of compounds, selected for their inability to combineirreversibly with thiols, are disclosed for use as NADH or NADPHmediators. The structural, electronic, and steric characteristics ofthese mediators render them nearly incapable of reacting with thiols.Because these mediators are virtually precluded from bindingirreversibly to the active site sulphydryl groups of NAD- andNADP-dependent dehydrogenases, inactivation of the enzyme and consequentloss of biosensor stability is circumvented.

[0044] The NADH and NADPH mediators can be used in the manufacture ofamperometric enzyme sensors for an analyte, where the analyte is asubstrate of an NAD- or NADP-dependent enzyme present in the sensor,such as those of the kind described in EP 125867-A. Accordingly,amperometric enzyme sensors of use in assaying for the presence of ananalyte in a sample, especially an aqueous sample, can be made. Forexample, the sample can be a complex biological sample such as abiological fluid (e.g., whole blood, plasma, or serum) and the analytecan be a naturally occurring metabolite (e.g., glucose,D-3-hydroxybutyrate, ethanol, lactate, or cholesterol) or an introducedsubstance such as a drug.

[0045] Of particular utility for the manufacture of amperometric enzymesensors, the present invention further provides an ink that includes theNADH and NADPH mediators disclosed herein.

[0046] The present invention also includes any precursor, adduct, orreduced (leuco) form of the above mediators that can be converted insitu by oxidation or decomposition to the corresponding activemediators. Such precursors or adducts can include hemiacetals,hemithioacetals, cyclic acetals, metal o-quinone complexes, protonatedforms, acetone adducts, etc.

[0047] A non-limiting list of enzymes that can be used in conjunctionwith the new mediators is provided in Table 1. TABLE 1 1.1.1.1 AlcoholDehydrogenase 1.1.1.27 Lactate Dehydrogenase 1.1.1.31 3-HydroxybutyrateDehydrogenase 1.1.1.49 Glucose-6-phosphate Dehydrogenase 1.1.1.47Glucose Dehydrogenase 1.2.1.46 Formaldehyde Dehydrogenase 1.1.1.37Malate Dehydrogenase 1.1.1.209 3-hydroxysteroid Dehydrogenase

[0048] Amperometric enzyme sensors adopting the mediators of the presentinvention generally use a test element, for example, a single-use strip.A disposable test element can carry a working electrode, for example,with the test reagents including the enzyme, the nicotinamide cofactor(i.e., NAD⁺ or NADP⁺), the mediators of the present invention forgeneration of a current indicative of the level of analyte, and areference/counter electrode. The test reagents can be in one or moreink-based layers associated with the working electrode in the testelement. Accordingly, the sensor electrodes can, for example, include anelectrode area formed by printing, spraying, or other suitabledeposition technique.

[0049] Referring to FIGS. 1 and 2, an electrode support 1, typicallymade of PVC, polycarbonate, or polyester, or a mixture of polymers(e.g., Valox, a mixture of polycarbonate and polyester) supports threeprinted tracks of electrically conducting carbon ink 2, 3, and 4. Theprinted tracks define the position of the working electrode 5 onto whichthe working electrode ink 16 is deposited, the reference/counterelectrode 6, the fill indicator electrode 7, and contacts 8, 9, and 10.

[0050] The elongated portions of the conductive tracks are respectivelyoverlaid with silver/silver chloride particle tracks 11, 12, and 13(with the enlarged exposed area 14 of track 12 overlying the referenceelectrode 6), and further overlaid with a layer of hydrophobicelectrically insulating material 15 that leaves exposed only positionsof the reference/counter electrode 14, the working electrode 5, the fillindicator electrode 7, and the contact areas 8, 9, and 10. Thishydrophobic insulating material serves to prevent short circuits.Because this insulating material is hydrophobic, it can serve to confinethe sample to the exposed electrodes. A suitable insulating material isSericard, commercially available from Sericol, Ltd. (Broadstairs, Kent,UK). Optionally, a first mesh layer 17, a second insulative layer 18, asecond mesh layer 19, a third insulative layer 20, and a tape 21 canoverlay the hydrophobic insulating material.

[0051] Respective ink mixtures can be applied onto a conductive track ona carrier, for example, in close proximity to a reference electrode 14connected to a second track. In this way, a sensor can be produced,which is capable of functioning with a small sample of blood or otherliquid covering the effective electrode area 5. The mixtures arepreferably, but not exclusively, applied to the carrier by screenprinting.

[0052] In general, NAD(P)-dependent dehydrogenases catalyze reactionsaccording to the equation:

RH₂+NAD(P)⁺→R+NAD(P)H+H⁺

[0053] where RH₂ represents the substrate (analyte) and R the product.In the process of the forward reaction, NAD(P)⁺ (i.e., NAD⁺ or NADP⁺) isreduced to NAD(P)H. Suitable amperometric biosensors provide anelectrochemical mediator that can reoxidize NAD(P)H, therebyregenerating NAD(P)⁺. Reoxidation occurs at an electrode to generate acurrent that is indicative of the concentration of the substrate.

[0054] In one embodiment, a dry sensor is provided. The sensor includesan elongated electrically insulating carrier having a pair oflongitudinal, substantially parallel, electrically conducting tracksthereupon, each track being provided at the same end with means forelectrical connection to a read-out and provided with an electrode, oneof the electrodes being the reference/counter electrode and the otherbeing the working electrode, together with test reagents. The sensor canbe configured in the form of a supporting strip of electricallyinsulating carrier material such as a synthetic polymer (e.g., PVC,polycarbonate, or polyester, or a mixture of polymers such as Valox)carrying the two electrodes supported on electrically conductive tracksbetween its ends. For example, the electrodes can take the form of tworectangular areas side by side on the carrier strip, as shown in FIG. 2(i.e., electrodes 14 and 16). Such areas can be designed as a targetarea to be covered by a single drop of sample, such as whole blood, fortesting the analyte. If desired, non-rectangular areas (e.g.,diamond-shaped, semicircular, circular, or triangular areas) can beemployed to provide a target area for optimized contact by a liquidsample.

[0055] The carrier includes at least two electrodes, namely areference/counter electrode and a working electrode. Other electrodessuch as a dummy electrode can also be included. These other electrodescan be of similar formulation to the working electrode (i.e., with theassociated test reagents), but lacking one or more of the workingelectrode's active components. A dummy electrode, for example, canprovide more reliable results, in that if charge passed at the dummyelectrode is subtracted from charge passed at the working electrode,then the resulting charge can be concluded to be due to the reaction ofinterest.

[0056] A membrane can be provided at or above the target to perform afiltration function. For example, a membrane can filter blood cells froma sample before the sample enters the test strip. Examples ofcommercially available membranes that can be used include Hemasep V,Cytosep, and Hemadyne (Pall Biosupport, Fort Washington, N.Y. 11050). Asan alternative, a filtration or cellular separation membrane can be castin situ. This can be achieved by casting hydrophobic polymers such ascellulose acetate, polyvinyl butyral and polystyrene and/or hydrophilicpolymers such as hydroxypropyl cellulose, polyvinylpyrrolidone,polyvinyl alcohol and polyvinyl acetate.

[0057] In another embodiment, there is provided a single use disposableelectrode strip for attachment to signal readout circuitry of a sensorsystem. The strip can detect a current representative of an analyte in aliquid mixture. The strip includes an elongated support adapted forreleasable attachment to the readout circuitry; a first conductorextending along the support and including a conductive element forconnection to the readout circuitry; a working electrode on the strip incontact with the first conductor and positioned to contact the mixture;a second conductor extending along the support, comprising a conductiveelement for connection to the readout circuitry; and a reference/counterelectrode in contact with the second conductive element and positionedto contact the mixture and the second conductor as depicted in FIG. 1.

[0058] The working electrode can include a printed layer on the support,and the printed layer itself can include an NAD or NADP-dependentdehydrogenase enzyme capable of catalyzing a reaction involving asubstrate for the enzyme. This layer can also include the correspondingnicotinamide cofactor and a mediator of the present invention capable oftransferring electrons between the enzyme-catalyzed reaction and thefirst conductor via NADH or NADPH, to create a current representative ofthe activity of both the enzyme and the analyte.

[0059] The first conductive element and the active electrode can bespaced apart from the second conductive element and thereference/counter electrode, and the electrodes sized and positioned topresent a combined effective area small enough to be completely coveredby a drop of blood or other test sample; typically the reaction zone is5 mm² but can be as large as 25 mm². The test sample completes anelectrical circuit across the active electrode and the reference/counterelectrode for amperometric detection of the activity of the enzyme.

[0060] In a preferred embodiment of the present invention a workingelectrode is produced by using a formulation which includes not only theenzyme, nicotinamide cofactor and the mediator but also filler andbinder ingredients which cause the working electrode to give anincreasing monotonic response to concentrations of interest for theanalyte being sensed when measured in a kinetic mode in which oxidationand reduction of the mediator both occur during the measurement. Theconcept is to provide a stable reaction layer on the surface of theworking electrode when the sample is applied. This allows the use ofmediators which are sparingly soluble in the sample. As the mediator isreduced by reaction with the enzyme, cofactor and analyte, it isretained in close proximity to the electrode surface so that it can bereadily reoxidized without significant loss to precipitation. Themaintenance of this thin reaction layer also allows the overallanalytical reaction to occur in a small volume of the overall sample soin effect what is measured is the flux of analyte from the bulk specimento this reaction layer.

[0061] This reaction layer needs to remain stable for at least the timeto conduct a reproducible kinetic measurement. Typical times for such ameasurement range between about 5 and 60 seconds, although stability forlonger times are preferred. Typically, the disposable electrode stripsof interest are mass produced and therefore it is desirable to have asafety margin with regard to any required property to account for theinherent variability in any mass manufacturing process.

[0062] The stability of the reaction layer can be improved by a propercombination of fillers and binders. The layer is preferably sufficientlystable to give an approximately linear reproducible response in akinetic measurement over the concentration range of interest for a givenanalyte. For instance, for Ketone bodies (measured as hydroxybutyrate)this would be between about 1 and 8 mM while for glucose it would bebetween about 2 and 40 mM.

[0063] The kinetic measurement involves the cycling of the mediatorbetween an oxidized state and a reduced state. The rate of this cycling,which is reflected in the current observed during the course of thetest, is dependent upon the concentration of the analyte in the sample.The greater the concentration of the analyte the more enzyme cofactorwhich is reduced in the course of the enzyme oxidizing the analyte. Themediator in turn becomes reduced in reoxidizing the cofactor and is thenreoxidized at the electrode surface. However, because of its very lowsolubility only a small amount of mediator is immediately available toreact with the reduced cofactor. Consequently mediator which reacts withreduced cofactor and is reoxidized at the electrode will then react withfurther reduced cofactor and this continues through the course of akinetic measurement. Thus the greater the concentration of the reducedcofactor (reflective of a greater concentration of analyte in thesample) the greater the driving force for the cycling of the mediatorand thus the greater the rate of cycling.

[0064] In some cases the cofactor may also engage in cycling between anoxidized state and a reduced state during the kinetic measurement. Thisdepends upon whether there is a sufficient quantity of cofactorinitially present to convert all the analyte present in the reactionlayer. If there is insufficient cofactor initially present as oxidizedcofactor is regenerated it promotes the oxidation of any analyteremaining in the reaction layer by becoming reduced again.

[0065] However, what is critical is that a given concentration ofanalyte reproducible results in the production of the same signal in thekinetic test for a particular electrode strip design and that the signalincreases monotonically, preferably linearly, with the concentration ofthe analyte (in other words that the signal be a true function of theanalyte concentration) over the concentration range of interest. Thisallows the manufacturer of the electrode strips to establish a universalcalibration for a given lot of electrode strips such that any givensignal obtained from a given strip under standard test conditionsuniquely correlates to a particular analyte concentration. Thus it isimportant that within the concentration range of interest there be nouncontrollable variable other than the analyte concentration which wouldsubstantially effect the signal.

[0066] The signal may be the current observed at a fixed time after thetest is initiated or it may be the current integrated over some periodoccurring some fixed time after the test is initiated (in essence thecharge transferred over some such period). The test is conducted bycovering the working electrode and a reference/counter electrode withsample and then applying a potential between them. The current whichthen flows is observed over some time period. The potential may beimposed as soon as the sample covers the electrodes or it may be imposedafter a short delay, typically about 3 seconds, to ensure good wettingof the electrodes by the sample. The fixed time until the current orcurrent integration is taken as the signal should be long enough toensure that the major variable affecting the observed current is theanalyte concentration.

[0067] The reference electrode/counter electrode may be a classicsilver/silver chloride electrode but it may also be identical to theworking electrode in construction. In one embodiment the two separateconductive tracks may both be coated with an appropriate formulation ofenzyme, cofactor and mediator in a binder and filler containing aqueousvehicle to yield a coating. In those cases in which the coating isnon-conductive, e.g. when the filler is a non-conductor, a commoncoating may overlay both electrodes. When a potential is applied one ofthe electrodes will function as a reference/counter electrode byabsorbing the electrons liberated at the other, working, electrode. Themediator at the reference/counter electrode will simply become reducedas a result of interaction with the electron flow at its electrode.

[0068] The reaction layer which yields the desired behavior is obtainedby formulating the working electrode with binder and filler ingredients.The object is to allow the sample to interact with the enzyme, cofactorand mediator but to also ensure that these chemically active ingredientsremain in the immediate vicinity of the surface of the electrode. Thebinder ingredient should include materials which readily increase theviscosity of aqueous media and promote the formation of films or layers.Typical of such materials are the polysaccharides such as guar gum,alginate, locust bean gum, carrageenan and xanthan. Also helpful arematerials commonly known as film formers such as polyvinyl alcohol(PVA), polyvinyl pyrrole, cellulose acetate, carboxymethyl cellulose andpoly (vinyl oxazolidinone). The filler ingredient should be aparticulate material which is chemically inert to the oxidationreduction reactions involved in the measurement and insoluble in aqueousmedia It may be electrically conductive or non-conductive. Typical,materials include carbon, commonly in the form of graphite, titaniumdioxide, silica and alumina.

[0069] The active electrode may be conveniently produced by formulatingthe enzyme, cofactor, mediator and binder and filler ingredients into anaqueous vehicle and applying it to the elongated, electricallyinsulating carrier having conducting tracks. The formulation may beapplied by printing such as screen printing or other suitabletechniques. The formulation may also include other ingredients such as abuffer to protect the enzyme during processing, a protein stabilizer toprotect the enzyme against denaturation and a defoaming agent. Theseadditional ingredients may also have an effect on the properties of thereaction layer.

[0070] The working electrode typically has a dry thickness between about2 and 50 μm; preferably between about 10 and 25 μm. The actual drythickness will to some extent depend upon the application technique usedto apply the ingredients which make up the working electrode. Forinstance thicknesses between about 10 and 25 μm are typical for screenprinting.

[0071] However, the thickness of the reaction layer is not solely afunction of the dry thickness of the working electrode but also dependsupon the effect of the sample on the working electrode. In the case ofaqueous samples the formulation of the working electrode ingredientswill effect the degree of water uptake this layer displays.

[0072] The filler typically makes up between about 20 and 30 weightpercent of the aqueous vehicle. The amounts of the other ingredients aretypically less than about 1 weight percent of the aqueous vehicle andare adjusted empirically to achieve the desired end properties. Forinstance, the amount of buffer and protein stabilizer are adjusted toachieve the desired degree of residual enzyme activity. In this regardone may use more enzyme and less stabilizer or less enzyme and morestabilizer to achieve the same final level of enzyme activity. Theamount of binder and defoaming agent should be adjusted to give suitableviscosities for the method of application with higher viscosities beingsuitable for screen printing and lower viscosities being suitable forrotogravure printing.

[0073] A suitable aqueous ink formulation can be formulated inaccordance with Table 2 with the balance being deformer, buffer, enzymeactivity enhancers and water to make up 1 gram of formulated ink. TABLE2 Enzyme (such as Glucose Dehyrogenase 200 to 4000 Units or3-hydroxybutyrate Dehydrogenase) Nicotinamide cofactor (such as NAD) 5to 30 weight percent Mediator (such as 1, 10 phenanthroline 0.1 to 1.5weight percent quinone) Filler (such as ultra fine carbon or 10 to 30weight percent titania) Binder (such as alginate or guar gum) 0.01 to0.5 weight percent Protein stabilizer (such as Trehalose 0.01 to 2weight percent or Bovine Serum Albumin)

[0074] The stability of the reaction layer can be readily evaluatedusing cyclic voltammetry with various time delays. The working electrodeformulation is evaluated by exposing it to a sample containing arelatively high concentration of analyte and subjecting it to a steadilyincreasing potential to a maximum value and then a steadily decreasingpotential back to no applied potential. The resulting current increasesto a peak value and then drops off as the voltage sweep continues. Suchcyclic voltammetry evaluations are conducted after various delay periodsafter the working electrode is exposed to the sample. The change in peakcurrent with increasingly long delay periods is a measure of thestability of the reaction layer. The more stable the reaction layer thesmaller the decrease in peak current.

[0075] An evaluation was conducted to compare the stability of a workingelectrode formulated in accordance with the teachings of the presentinvention to that of a “working electrode” formulated according to theteachings of Geng et al. at pages 1267 to 1275 of Biosensors andBioelectronics, Volume II, number 12 (1996). The working electroderepresentative of the present invention was formulated with about 25weight percent filler (ultra fine carbon), binder, protein stabilizerand deformer as taught hereinabove and the working electroderepresentative of Geng was formulated with a high molecular weight poly(ethylene oxide) as described at page 1267 of the Geng article. In eachcase a potential was applied at a scan rate of 50 millivolt per secondup to 400 mV versus a silver/silver chloride reference electrode afterexposing the working electrode to a 20 mM aqueous solution of glucosefor 3 seconds and 60 seconds. The formulation according to the presentinvention yields a stable reaction layer in which the peak current after60 seconds is 60% of that observed after 3 seconds while the formulationaccording to the Geng article yields an unstable reaction layer in whichno peak current is observable after 60 seconds exposure.

[0076] This is attributed to a dissolution of the electrode with a lossof the reagents to the bulk solution. The respective voltammograms areas follows:

[0077] The test strips of this invention can detect analytes that aresubstrates of NAD- or NADP-dependent dehydrogenase enzymes using amediator selected from the compounds disclosed herein, such as 1,10-PQ.

[0078] Test strips according to this invention are intended for use withelectronic apparatus and meter systems. These control the progress ofthe electrochemical reaction (e.g., by maintaining a particularpotential at the electrodes), monitor the reaction, and calculate andpresent the result. A particular feature that is desirable in a metersystem for use with test strips of this type is the capability ofdetecting the wetting of the reaction zone by sample fluid, thusallowing timely initiation of the measurement and reducing the potentialfor inaccuracies caused by user error. This goal can be achieved byapplying a potential to the electrodes of the test strip as soon as thestrip is inserted into the meter; this potential can be removed for ashort time to allow wetting to be completed before initiation ofmeasurement.

[0079] The meter can also feature a means for automatically identifyingtest strips for measuring different analytes. This can be achieved, forexample, when one or more circuit loops are printed on the test strip;each loop can provide a resistance characteristic of the type of strip,as described in U.S. Pat. No. 5,126,034 at column 4, lines 3 to 17. As afurther alternative, notches or other shapes might be cut into theproximal end of the test strip; switches or optical detectors in themeter can detect the presence or absence of each notch. Other strip-typerecognition techniques include varying the color of the strips andproviding the meter with a photodetector capable of distinguishing therange of colors; and providing the strips with barcodes, magneticstrips, or other markings, and providing the meter with a suitablereading arrangement.

[0080] In one example of a test strip for large scale production, thestrip electrodes have a two-electrode configuration comprising areference/counter electrode and a working electrode. The carrier can bemade from any material that has an electrically insulating surface,including poly(vinyl chloride), polycarbonate, polyester, paper,cardboard, ceramic, ceramic-coated metal, blends of these materials(e.g., a blend of polycarbonate and polyester), or another insulatingsubstance.

[0081] A conductive ink is applied to the carrier by a deposition methodsuch as screen printing. This layer forms the contact areas, which allowthe meter to interface with the test strip, and provides an electricalcircuit between the contacts and the active chemistry occurring on thestrip. The ink can be an air-dried, organic-based carbon mixture, forexample. Alternative formulations include water-based carbon inks andmetal inks such as silver, gold, platinum, and palladium. Other methodsof drying or curing the inks include the use of infrared, ultraviolet,and radio-frequency radiation.

[0082] A layer forming the reference/counter electrode is printed withan organic solvent-based ink containing a silver/silver chloridemixture. Alternative reference couples include Ag/AgBr, Ag/AgI, andAg/Ag₂O. The print extends to partially cover the middle track of thecarbon print where it extends into the reaction zone. It is useful ifseparate parts of this print are extended to cover parts of other carbontracks outside the reaction zone, so that the total electricalresistance of each track is reduced.

[0083] A layer of dielectric ink can optionally be printed to cover themajority of the printed carbon and silver/silver chloride layers. Inthis case, two areas are left uncovered, namely the electrical contactareas and the sensing area which will underlie the reactive zone asdepicted in FIGS. 1 and 2. This print serves to define the area of thereactive zone, and to protect exposed tracks from short circuit.

[0084] For the working electrode, one or more inks are deposited to aprecise thickness within a defined area on top of one of the conductivetracks within the reaction zone, to deposit the enzyme, cofactor and amediator of the present invention. It is convenient to do this by meansof screen printing. Other ways of laying down this ink include inkjetprinting, volumetric dosing, gravure printing, flexographic printing,and letterpress printing. Optionally, a second partially active ink canbe deposited on a second conductive track to form a dummy electrode.

[0085] Polysaccharides can optionally be included in the inkformulation. Suitable polysaccharides include guar gum, alginate, locustbean gum, carrageenan and xanthan. The ink can also include a filmformer; suitable film-forming polymers include polyvinyl alcohol (PVA),polyvinyl pyrrole, cellulose acetate, CMC, and poly(vinyloxazolidinone). Ink fillers can include titanium dioxide, silica,alumina, or carbon.

[0086] The following are illustrative, non-limiting examples of thepractice of the invention:

EXAMPLE 1

[0087] Mediators:

[0088] Meldola's Blue (MB) (Compound 3) was obtained as the hemi-ZnCl₂salt from Polysciences, Inc. 2,6-Dichloroindophenol (DCIP) (Compound 6)and Tris buffer were purchased from Sigma. The phosphate buffered saline(PBS) solution (Dulbecco's formula) was prepared from tablets suppliedby ICN Biomedicals. Ltd.

[0089] D-3-Hydroxybutyrate dehydrogenase (HBDH; EC 1.1.1.30) fromPseudomonas sp. was purchased from Toyobo Co., Ltd. p-Nicotinamideadenine dinucleotide (NAD⁺) and D,L-3-hydroxybutyric acid were suppliedby Boehringer Mannheim.

[0090] 1,10-Phenanthroline quinone (1,10-PQ) (Compound 7) was preparedaccording to the method of Gillard et al. (J. Chem. Soc. A, 1447-1451,1970). 1,7-Phenanthroline quinone (1,7-PQ) (Compound 8) was synthesizedusing the procedure described by Eckert et al. (Proc. Natl. Acad. Sci.USA, 79:2533-2536, 1982). 2,9-Dimethyl-1,10-phenanthroline quinone(2,9-Me₂-1,10-PQ) (Compound 10) was synthesized as a byproduct of thenitration of neocuproine as disclosed by Mullins et al. (J. Chem. Soc.,Perkin Trans. 1, 75-81, 1996). 1-Methoxy phenazine methosulphate(1-MeO-PMS) (Compound 5) was prepared via the methylation of 1-methoxyphenazine adapted from the method described by Surrey (Org Synth. Coll.Vol. 3, Ed. E. C. Horning, Wiley, New York, 753-756). 1-Methoxyphenazine was synthesized by a modified Wohl-Aue reaction as reported byYoshioka (Yakugaku Zasshi, 73:23-25, 1953). 4-Methyl-1,2-benzoquinone(4-Me-BQ) (Compound 4) was prepared via oxidation of 4-methyl catecholwith o-chloranil according to a general procedure by Carlson et al. (J.Am. Chem. Soc., 107:479485, 1985). The 1,10-PQ complex[Ru(bpy)₂(1,10-PQ)](PF₆)₂ (Compound 12) was obtained from [Ru(bpy)₂Cl₂](Strem Chemicals, Inc.) as reported by Goss et al. (Inorg. Chem.,24:4263-4267, 1985).

[0091] Preparation of 1-Me-1,10-phenanthrolinium quinonetrifluoromethane sulphonate (1-Me-1,10-PQ⁺) (Compound 11):

[0092] Methyl trifluoromethane sulphonate (Aldrich) (1.0 ml) was addedto a solution of 1,10-PQ (0.50 g, 2.38 mmol) in anhydrous methylenechloride (25 ml) under nitrogen. Immediate precipitation occurred andthe resulting mixture was stirred for 24 hours. Filtration followed bywashing with methylene chloride afforded l-Me-1,10-PQ⁺ (0.65 g, 73%) asa fine yellow powder.

[0093] Evaluation of Meldola's Blue and 1,10-PQ as NADH Mediators in DryStrips:

[0094] Screen-printed electrodes incorporating 1,10-PQ and MB wereproduced from an organic carbon ink containing these NAD(P)H mediatorsat a level of 3.5 mg/g ink. The solid mediators were mixed into acommercial conducting carbon ink (Gwent Electronic Materials).

[0095] The dose response curve for the electrodes containing 1,10-PQtested with aqueous NADH solutions (0-16.7 mM) in PBS at a poisepotential of +400 mV versus a printed Ag/AgCl reference electrode isshown in FIG. 3. A slope of 0.58 μA mM⁻¹ NADH was recorded. The doseresponse curve for the electrodes containing MB tested with aqueous NADHsolutions (0-12.4 mM) at a poise potential of +100 mV versus a printedAg/AgCl reference electrode is shown in FIG. 4. An increased slope of8.48 μA mM⁻¹ NADH was observed.

[0096] Assessment of Mediator Inhibition of D-3-HydroxybutyrateDehydrogenase:

[0097] A series of 18 solutions (2.5 ml each) were prepared, eachcontaining 50 U/ml HBDH and 1.29 or 2.58 mg of the following NAD(P)Hmediators: MB(3), 4-Me-BQ(4), 1-MeO-PMS(5), DCIP(6), 1,10-PQ(7),1,7-PQ(8), 2,9-Me₂-1,10-PQ(10), 1-Me-1,10-PQ⁺(11), and[Ru(bpy)₂(1,10-PQ)](PF₆)₂ (12) in Tris buffer (50 mM, pH 8.2). A controlsolution was also prepared, containing enzyme but no mediator. Thesolutions were incubated for 0.5 hours at 37.5 C, then assayed (intriplicate) for NADH at 340 nm, using a Sigma DiagnosticsD-3-hydroxybutyrate kit. The extent of the interference of the addedmediator with the assay rate compared to the control afforded aquantitative measure of the mediator's efficiency as an oxidant of NADH.

[0098] The enzyme was then reisolated from the mediator solutions byfiltration through a polysulfone membrane (nominal molecular weightcut-off: 30,000) in a microcentrifuge filter (Millipore). The enzymeremaining on the filter was dissolved in Tris buffer (0.2 ml), and theresulting solution was assayed (in triplicate) with the Sigma kit. Bycomparing the results of the assays before and after filtration, theeffect of any covalently and/or irreversibly bound mediator on theenzyme activity could be determined.

[0099] The results of the two assays on each solution before and afterfiltration are collected in Table 3. TABLE 3 Assay Rate (absorbanceunits/min) Mediator control before after (Compound No.) (no mediator)filtration filtration 1,10-PQ 0.167 0.149 0.160(96%) 1,7-PQ 0.155 0.1150.150(97%) MB 0.167 0.008 0.026(16%) 4-Me-BQ 0.170 0.005 0.007(4%)1-MeO-PMS 0.150 0.009 0.071 (47%) DCIP 0.150 0.104 0.085 (57%)2,9-Me₂-1,10-PQ 0.197 0.189 n/a 1-Me-1,10-PQ* 0.197 0.150 0.185 (94%)[Ru(bpy)₂(1,10-PQ)](PF₆)₂ 0.197 0.114 0.193 (98%)

[0100] Although these results demonstrated that the phenanthrolinequinone mediators were relatively inefficient NADH mediators compared toMeldola's Blue and 1-MeO-PMS (i.e., the assay rate “before filtration”was depressed only to a small extent), over 90% of the original enzymeactivity for the solutions containing 1,10-PQ, 1,7-PQ, 1-MeO-1,10-PQ, or[Ru(bpy)₂(1,10-PQ)](PF₆)₂ was restored “after filtration.” This was notthe case for MB, 1-MeO-PMS, DCIP, or 4-Me-BQ. Indeed, the quinonemediator 4-Me-BQ proved to be the most potent inhibitor with only 4% ofthe original activity remaining “after filtration.” Thus, the latterfour mediators partially inactivate HBDH while the newly describedmediators advantageously had little or no effect on enzyme activity.

[0101] The percentage residual enzyme activities for each mediator aredisplayed as a bar chart in FIG. 5, which reveals that the mediators ofthe present invention, represented by black bars, are not stronginhibitors of HBDH. In contrast, MB, 4-Me-BQ, 1-MeO-PMS, and DCIP allirreversibly inhibited HBDH, with concomitant losses in activity rangingfrom 43 to 96%; these results are represented by grey bars in FIG. 5.

EXAMPLE 2

[0102] Evaluation of Meldola's Blue and 1,10-PQ in Dry Strips containingHBDH:

[0103] Screen-printed electrodes were produced from an aqueous carbonink incorporating 1,10-PQ or MB at a level of 2.4 or 4.3 mg/g ink,respectively, together with the enzyme HBDH (120 units/g ink) and NAD⁺(110 mg/g ink). The ink also contained a polysaccharide binder.

[0104] The dose response curves for the electrodes containing 11,10-PQare given in FIG. 6. The electrodes were tested after 4,14, and 26 weeksof storage (30° C., desiccated) with aqueous D-3-hydroxybutyratesolutions (0-25 mM) in PBS at a poise potential of +400 mV versus aprinted Ag/AgCl reference electrode. All three dose responses werenon-linear and levelled out with a current of 8.5 μA being recorded at24 mM D-3-hydroxybutyrate. This demonstrated that the response of thedry electrodes was stable for at least 26 weeks.

[0105] The dose response curves for the electrodes containing MB areprovided in FIG. 7. The electrodes were tested after 2 and 14 weeksstorage (30° C., desiccated) with aqueous D-3-hydroxybutyrate solutions(0-28 mM) in PBS at a poise potential of +100 mV versus a printedAg/AgCl reference electrode. The dose response curves were similar tothose in FIG. 4. A current of 8.6 μA was recorded at 24 mMD-3-hydroxybutyrate for these electrodes after 2 weeks storage. This isalmost identical to responses obtained from dry strips containing1,10-PQ.

[0106] This result demonstrated that the ability of a compound such asMB to mediate very efficiently with NADH compared to 1,10-PQ isoutweighed by the fact that it inhibits HBDH. Furthermore, the stabilityof the electrode response to D-3-hydroxybutyrate is compromised throughthe inactivation of HBDH by MB. FIG. 7 shows that the response of theseelectrodes dropped by an unacceptable margin of approximately 7% after14 weeks storage.

[0107] In summary, biosensor electrodes containing a mediator of thepresent invention displayed responses which were stable after at least26 weeks storage. In contrast, those electrodes incorporating atraditional mediator such as MB which is an irreversible enzymeinhibitor exhibited responses which declined after only 14 weeksstorage.

EXAMPLE 3

[0108] Evaluation of 1,10-PQ in Dry Strips Containing GlucoseDehydrogenase (GDH):

[0109] Screen-printed electrodes were produced from an aqueous carbonink incorporating 1,10-PQ or MB at a level of 2.4 or 4.3 mg/g ink,respectively, together with the enzyme Glucose dehydrogenase (120units/g ink) and NAD⁺ (110 mg/g ink). The ink also contained apolysaccharide binder.

[0110] The calibrated dose response curve for the electrodes is given inFIG. 8. The electrodes were tested with whole blood containingphysiologically relevant concentrations of glucose ranging from 3.3 to26 mM. A poise potential of +50 mV was maintained against a printedAg/AgCl electrode. The electrodes produced a linear response over theglucose range. Thus, it was demonstrated that a mediator of the presentinvention can be used to construct a clinically useful glucose sensorwhich operates at a particularly low applied potential.

EXAMPLE 4

[0111] Electrode strips were prepared utilizing the constructionillustrated in FIGS. 1 and 2 with a silver/silver chloridereference/counter electrode and a working electrode prepared by screenprinting a formulation in accordance with Table 2. In one case, thefiller was 25 weight percent ultra fine carbon and in the other case thefiller was 25 weight percent titania. In both cases the enzyme wasGlucose Dehydrogenase (GDH), the cofactor was NAD, the mediator was1,10-PQ, the binder was guar gum, the protein stabilizer was Bovineserum albumin (BSA) and the buffer was Tris (0.325 weight percent).

[0112] These electrode strips were evaluated by applying a 200 mVpotential between the reference/counter electrode and the workingelectrode while an aqueous glucose solution covered both electrodes. Theobserved current from 15 to 20 seconds after the application of thepotential was integrated and plotted against the glucose contents of thetest solutions. The carbon-filled formulation gave a slope of 2.6microcoulomb per mM of glucose and an X axis intercept of −1microcoulomb while the titania-filled formulation gave a slope of 1.5microcoulomb per mM of glucose and an X axis intercept of 0.6microcoulomb. The plots were as follow:

Other Embodiments

[0113] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not to limit thescope of the invention. Other aspects, advantages, and modifications arewithin the scope of the invention.

1 A single use disposable electrode strip for attachment to the signalreadout circuitry of a sensor system to detect a current representativeof an analyte in an aqueous sample, the strip comprising: a) anelongated support having a substantially flat, planar surface, adaptedfor releasable attachment to said readout circuitry; b) a firstconductor extending along said surface and comprising a conductiveelement for connection to said readout circuitry;  an active electrodeon said surface in contact with said first conductor, said activeelectrode comprising a nicotinamide co-factor-dependent enzyme, anicotinamide cofactor, and a mediator compound having one of the twofollowing formulae:

 where X and Y can independently be oxygen, sulphur, CR³R⁴, NR³, orNR³R⁴ or the functional group CZ¹Z², where Z¹ and Z² are electronwithdrawing groups; R₁ and R₂ can independently be a substituted orunsubstituted aromatic or heteroaromatic group; and R³ and R⁴ canindependently be a hydrogen atom, a hydroxyl group or a substituted orunsubstituted alkyl, aryl, heteroaryl, amino, alkoxyl, or aryloxyl groupc) a second conductor extending along said surface, comprising aconductive element for connection to said readout circuitry; d) areference/counter electrode in contact with said second conductor; e) athird conductor extending along said surface, comprising a conductiveelement for connection to readout circuitry; f) a fill indicatorelectrode in contact with said third conductor; g) said conductors beingspaced apart so as not to be in electrical contact and being configuredso as not to be brought into electrical contact when said aqueous sampleis placed on said strip; h) said active electrode and saidreference/counter electrode being configured so that both may besimultaneously covered by a small drop of said aqueous sample to providean electrical conduction path between said electrodes. 2 The electrodestrip of claim 1 wherein the mediator compound is 1,10-phenantholinequinone. 3 The electrode strip of claim 2 wherein the cofactor-dependentenzyme is Glucose Dehydrogenase. 4 The electrode strip of claim 1wherein the cofactor-dependent enzyme is 3-HydroxybutyrateDehydrogenase. 5 A process of measuring the concentration in an aqueoussample of an analyte subject to oxidation by a NAD(P)⁺ dependent enzymecomprising: a) providing the electrode strip of claim 1; b) applying thesample containing the analyte to the electrode strip; c) detecting thesample applied to the electrode strip: d) oxidizing the analyte with theNAD(P)⁺ dependent enzyme in the presence of NAD(P)⁺; oxidizing theNAD(P)H generated by reaction with the analyte and NAD(P)⁺ dependentenzyme with the mediator compound of step a); and e) applying anelectrical potential at an electrode to reoxidize the mediator compoundreduced in oxidizing NAD(P)H and observing the resultant current. 6 Theprocess of claim 5 wherein the NAD(P)⁺ dependent enzyme, NAD(P)⁺, andmediator compound have been applied to the surface of said electrode incombination with a binder and a filler. 7 The process of claim 6 whereinthe current observed during the measurement period is linearly relatedto the concentration of the analyte in the sample. 8 The process ofclaim 5 wherein the mediator is 1,10-phenanthroline quinone. 9 Theprocess of claim 8 wherein the cofactor-dependent enzyme is GlucoseDehydrogenase. 10 The process of claim 8 wherein the cofactor dependentenzyme is 3-Hydroxybutyrate Dehydrogenase. 11 The process of claim 5wherein the applied potential is 200 mV or less. 12 The process of claim5, wherein the current is linearly related to the concentration of theanalyte. 13 The electrode strip of claim 1, wherein the mediator is4,7-phenanthroline quinone. 14 The electrode strip of claim 1, whereinthe mediator is 1,7-phenanthroline quinone. 15 The electrode strip ofclaim 1, further including a dummy electrode. 16 The electrode strip ofclaim 1, further including circuit loops for providing a resistancecharacteristic of the electrode strip to identify the test strip asbeing suitable for a particular analyte. 17 The electrode strip of claim1, further including at least one notch or other shape in an end of thetest strip to identify the test strip as being suitable for a particularanalyte. 18 The electrode strip of claim 1, further including at leastone switch to identify the test strip as being suitable for a particularanalyte. 19 The electrode strip of claim 1, further including at leastone optical detector to identify the test strip as being suitable for aparticular analyte. 20 A single use disposable electrode strip forattachment to the signal readout circuitry of a sensor system to detecta current representative of an analyte in an aqueous sample, the stripcomprising: a) an elongated support having a substantially flat, planarsurface, adapted for releasable attachment to said readout circuitry; b)a first conductor extending along said surface and comprising aconductive element for connection to said readout circuitry; c) anactive electrode on said surface in contact with said first conductor;d) a second conductor extending along said surface, comprising aconductive element for connection to said readout circuitry; e) areference/counter electrode in contact with said second conductor; f) athird conductor extending along said surface, comprising a conductiveelement for connection to readout circuitry; g) a fill indicatorelectrode in contact with said third conductor; h) said conductors beingspaced apart so as not to be in electrical contact and being configuredso as not to be brought into electrical contact when said aqueous sampleis placed on said strip; i) said active electrode and saidreference/counter electrode being configured so that both may besimultaneously covered by a small drop of said aqueous sample to providean electrical conduction path between said electrodes. 21 The electrodestrip of claim 20, further including circuit loops for providing aresistance characteristic of the electrode strip to identify the teststrip as being suitable for a particular analyte. 22 The electrode stripof claim 20, further including at least one notch or other shape in anend of the test strip to identify the test strip as being suitable for aparticular analyte. 23 The electrode strip of claim 20, furtherincluding at least one switch to identify the test strip as beingsuitable for a particular analyte. 24 The electrode strip of claim 20,further including at least one optical detector to identify the teststrip as being suitable for a particular analyte.